Method for inhibiting virus replication in mammalian cells using carbostyil derivatives

ABSTRACT

A method for inhibiting nucleoside and nucleobase transport in mammalian cells, as well as to a method for inhibition of virus replication, and augmenting phosphorylation of nucleoside analogues, are disclosed, wherein each method uses, as the active agent, a carbostyril derivative.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT10595/09141 filed Jul. 8, 1995 which isa continuation-in-part of U.S. patent application Ser. No. 08/283,707,filed Aug. 1, 1994, now U.S. Pat. No. 5,504,093.

FIELD OF THE INVENTION

The present invention relates to a method for inhibiting nucleoside andnucleobase transport in mammalian cells, as well as to a method forinhibition of virus replication, and augmenting phosphorylation ofnucleoside analogues, wherein each method uses, as the active agent, acarbostyril derivative.

BACKGROUND OF THE INVENTION I. Carbostyrils

Carbostyril derivatives represented by the following general formula(1), and salts thereof: ##STR1## wherein R is a benzoyl group which mayoptionally have lower alkoxy groups on the phenyl ring as substituentsand the carbon-carbon bond in the 3 and 4 positions of the carbostyrilskeleton is a single bond or double bond,

are well-known in the art (U.S. Pat. No. 4,415,572, which isincorporated by reference herein in its entirety).

These carbostyrils have been found to be an oral inotropic agent thataugments myocardial contractility in model systems, with little effecton the heart rate or myocardial oxygen consumption (Feldman et al, N.Engl. J. Med., 329:149-155 (1993)), and are useful for treatment ofpatients with congestive heart failure (U.S. Pat. No. 4,415,572; andHori et al, Jpn Circ. J., 50:659-666 (1986)). Several studies havedemonstrated that the above carbostyrils improve hemodynamic indexes,and exercise capacity in congestive heart failure patients (Inoue et al,Heart Vessels, 2:166-171 (1986); Sasayama et al, Heart Vessels, 2:23-28(1986); and Feldman et al, Am. Heart J., 116:771-777 (1988)). Inaddition, multi-center randomized placebo-controlled trials both inJapan and in the United States have demonstrated that these carbostyrilsimprove both quality of life and reduced the risk of death in patientswith congestive heart failure (OPC-8212 Multicenter Research Group,Cardiovasc. Drugs Ther., 4:419-425 (1990); Feldman et al, Am. J.Cardiol., 68:1203-1210 (1991); and Feldman et al, N. Engl. J. Med.,329:149-155 (1993)).

The mechanisms of action associated with the inotropic properties ofthese carbostyrils include a decrease in potassium current (Iijima etal, J. Pharmacol. Exp. Ther., 240:657-662 (1987)), a mild inhibition ofphosphodiesterase, and an increase in the inward calcium current (Yataniet al, J. Cardiovasc. Pharmacol., 13:812-819 (1989); and Taira et al,Arzneimittelforschung, 34:347-355 (1984)). However, the dose of thecarbostyrils which was most effective in reducing mortality (60 mgdaily) showed no or little hemodynamic effect, implying that the drugmay reduce mortality through another mechanism, rather than its positiveinotropic effect (Feldman et al, N. Eng. J. Med., 329:149-155 (1993);and Packer, N. Engl. J. Med., 329:201-202 (1993)).

The above carbostyrils are also known to inhibit the production ofvarious cytokines, including TNF-α and IL-6, bylipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMC)in a dose-dependent manner (Maruyama et al, Biochem. Biophys. Res.Commu., 195:1264-1271 (1993); and Matsumori et al, Circul., 89:955-958(1994)).

Moreover, they can induce a reversible neutropenia associated with adecrease in CFU-C (Feldman et al, Am. Heart J., 116:771-777 (1988);OPC-8212 Multicenter Research Group, Cardiovasc. Drugs, Ther., 4:419-425(1990); Feldman et al, Am. J. Cardiol., 68:1203-1210 (1991); and Feldmanet al, N. Engl. J. Med., 329:149-155 (1993)).

Additionally, the above carbostyrils have been found to be useful inregulating apoptosis (programmed cell, death), and in the treatment ofcancer, inhibition of tumor metastasis and inhibition of RNA virusreplication (U.S. patent application Ser. No. 07/989,028, filed Apr. 30,1993, which corresponds to European Patent Publication 0552373, each ofwhich is incorporated by reference herein in their entirety; Nakai etal, Jpn. J. Cancer Res., Abstract, and Proc. Jpn. Cancer Assoc., page581 (1993); and Maruyama et al, Biochem. Biophys. Res. Comm.,195:1264-1271 (1993)).

It has been surprising to find in the present invention that thesecarbostyrils, particularly the species 3,4-dihydro-6-4-(3,4-dimethoxybenzoyl)-1- piperazinyl!-2(1H)-quinoline (hereinafter"vesnarinone"), inhibit nucleoside and nucleobase transport in mammaliancells, as the structures of these compounds are entirely different fromthe structure of known compounds which inhibit nucleoside and nucleobasetransport.

II. Epstein-Barr Virus

Epstein-Barr virus (EBV), a human lymphotropic herpes group DNA virus,infects human B lymphocytes, and is linked to a variety oflymphoproliferative diseases (Miller et al, Virol., Second Edition,pages 1921-1958 (1990)). There are several reports indicating that thereactivation of EBV may be linked to the development of B-cell mediatedautoimmunity (Fox, J. Virol. Methods, 21:19-27 (1988); Fox et al,Springer Semin. Immunopathol., 13:217-231 (1991); and Logtenberg et al,Immunol. Rev., 128:23-47 (1992)).

EBV persists in B lymphocytes as a latent infection in vivo and invitro. In its latent form, the EBV genome exists in a circular episomalform, and gene expression is relatively limited (Sample et al, J.Virol., 64:1667-1974 (1990)). In vitro, the expression of latent genesis associated with immortalization and with resistance of host cells toapoptosis, related, at least in part, to the up-regulation of bcl-2 geneexpression (Henderson et al, Cell, 65:1107-1115 (1991); and Gregory etal, Nature, 349:612-614 (1991)). Replication of the EBV genome in thelatent phase is controlled by host cell polymerases, and occurs strictlyduring S-phase of the host cell cycle (Adams, J. Virol., 61:1743-1746(1987)).

Various reagents including phorbol esters, n-butyrate, halogenatedpyrimidines, calcium ionophores, and anti-immunoglobulin (Ig) antibodiescan initiate the switch into the lytic or productive EBV replicationphase in latently infected B lymphocytes (Tovey et al, Nature,276:270-272 (1978); Gerber, Proc. Natl. Acad. USA, 69:83-85 (1972);Faggioni et al, Science, 232:1554-1556 (1986); Takada, Int. J. Cancer,33:27-32 (1984); and Takagi et al, Virol., 185:309-315 (1991)).Replication of EBV genome in the productive phase, where a large numberof infectious EBV virions are produced, is independent of host cell DNAreplication, but can be affected by alteration of the intracellulardeoxynucleobase pool (Datta et al, Proc. Natl. Acad. Sci. USA,77:5163-5166 (1980)). Clinicopathologicaly, entry into the productivephase of the EBV cycle is purported to play an important role in thereactivation of EBV infection in vivo.

It has been surprising to find in the present invention that the abovecarbostyrils, particularly vesnarinone, are useful in inhibiting DNAvirus replication, as evidenced by data in an EBV model system, and datain a CMV model, and provides a synergistic effect, when used togetherwith an anti-DNA virus compound.

III. Augmentation of Phosphorylation of Nucleoside Analogues

It also had been surprisingly found in the present invention that theabove carbostyrils, particularly vesnarinone, inhibit transport ofthymidine, but not AZT, are useful in augmenting phosphorylation ofnucleoside analogues, particularly, AZT, and provide a synergisticeffect, when used together with an anti-RNA virus compound, ininhibiting RNA virus replication, as evidenced by data in an HIV modelsystem.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for inhibitingnucleoside and nucleobase transport in a mammalian cell.

Another object of the present invention is to provide a method forinhibiting DNA virus replication.

A further object of the present invention is to provide a method fortreatment of EBV infection.

A still further object of the present invention is to provide a methodfor treatment of CMV infection.

An additional object of the present invention is to provide a method foraugmenting phosphorylation of nucleoside analogues.

Yet another object of the present invention is to provide a method forinhibiting RNA virus replication.

A still further object of the present invention is to provide a methodfor treatment of HIV infection.

These and other objects of the present invention, which will be apparentfrom the detailed description of the invention provided hereinafter,have been met by the use of a carbostyril derivative represented by thefollowing general formula (1), and salts thereof: ##STR2## wherein R isa benzoyl group which may optionally have lower alkoxy groups on thephenyl ring as substituents and the carbon-carbon bond in the 3 and 4positions of the carbostyril skeleton is a single bond or double bond.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show ³ H!thymidine incorporation by human T cells stimulatedfor 48 hrs with 10 μg/ml PHA in the presence of 0-250 μM vesnarinone(FIG. 1A); and in the presence of RPMI 1640 medium, in the absence ofany drug, in the presence of HEPES buffer, and in the presence of 250 μMvesnarinone, 100 μM deferoxamine or 15 nM aphidicolin (FIG. 1B). FIG. 1Cshows cell numbers after human T cells are stimulated for 0-120 hrs with10 μg/ml PHA () in the presence of RPMI 1640 medium in the absence ofany drug (∘), in the presence of HEPES buffer (□), 250 μM vesnarinone(▪), 100 μM deferoxamine (Δ) or 15 nM aphidicolin (▴).

FIGS. 2A-2F show cellular DNA content, as measured by fluorescenceintensity, determined after human T cells were stimulated for 0-120 hrswith: RPMI 1640 medium (FIG. 2A); 10 μg/ml PHA (FIG. 2B); 10 μg/ml PHAin the presence of HEPES buffer (FIG. 2C); 10 μg/ml PHA in the presenceof 250 μM vesnarinone (FIG. 2D); 10 μg/ml PHA in the presence of 15 nMaphidicolin (FIG. 2E); or 10 μg/ml PHA in the presence of 100 μMdeferoxamine (FIG. 2F).

FIG. 3 shows ³ H!thymidine, ³ H!uridine and ³ H!amino acid incorporationby human T cells stimulated for 48 hrs with 10 μg/ml PHA in the presenceof 250 μM vesnarinone, 100 μM deferoxamine or 15 nM aphidicolin.

FIG. 4 shows the effects on ³ H!nucleoside or ³ H!nucleobase transportin human T cells stimulated for 48 hrs with 10 μg/ml PHA in the presenceof 250 μM vesnarinone, 100 μM deferoxamine, 15 nM aphidicolin or 1.0 μMdipyridamole.

FIGS. 5A-5D show the dose-dependent effects of 0-750 μM vesnarinone onthe transport of ³ H!adenosine (∘) or ³ H!adenine () in Raji cells(FIG. 5A) or CHO cells (FIG. 5C); and the dose-dependent effects of0-100 μM dipyridamole on the transport of ³ H!adenosine (∘) or ³H!adenine () in Raji cells (FIG. 5B) or CHO cells (FIG. 5D).

FIGS. 6A-6B show cell numbers counted 120 hrs after incubation of CHOcells with 50 nM 5-Fud in the presence of 0-250 μM vesnarinone (FIG.6A); or 1-10 μM dipyridamole (FIG. 6B).

FIG. 7 shows the actual percentage of DMSO-released Akata cellsstimulated with 50 μg/ml of anti-IgG antibody in the presence ofvesnarinone at concentrations ranging from 0-100 μg/ml, and whichexpress ZEBRA, as determined by flow cytometry (FCM), and the number ofcells positive for EBV DNA, as determined by in situ hybridization.

FIGS. 8A-8D represent Western blots of lysates from DMSO-released Akatacells stimulated with 50 μg/ml of anti-IgG antibody in the absence (-)or presence of vesnarinone at a concentration of 30 μg/ml (+). Theproducts of BZLF1 (ZEBRA) (FIG. 8A); BRLF1 (R) (FIG. 8B); BHRF1 (EA-R)(FIG. 8C); and BMRF1 (EA-D) (FIG. 8D) were assayed by Western blottingof lysates from the cells harvested 3 hrs and 20 hrs after stimulationwith anti-IgG.

FIGS. 9A-9F represent flow cytometry analysis of DMSO-released Akatacells stimulated with 50 μg/ml of anti-IgG for 20 hrs in the presence ofvesnarinone at concentrations ranging from 0-100 μg/ml. The controlshows the results of samples without anti-IgG stimulation. The 2C peakcorresponding to the DNA content of the G1 population appears aroundchannel numbers 75 to 100.

FIGS. 10A-10D show pmoles of dNTP extracted with 60% (v/v) ethanol from10⁶ logarithmically growing DMSO-arrested Akata cells, Akata cellsassayed 13 hrs and 21 hrs after DMSO-release without vesnarinone withoutanti-IgG stimulation (∘), or with IgG stimulation (□), and Akata cellsassayed 13 hrs and 21 hrs after DMSO-release with 100 μg/ml vesnarinonewithout (⋄) anti-IgG stimulation or with anti-IgG stimulation (x), andquantitated by an enzymatic method.

FIG. 11 shows the frequency of logarithmically growing Akata cellsdemonstrating productive replication of EBV when cultured in mediumcontaining 20 μg/ml vesnarinone for 7 days (pre-treatment), 24 hrs afterstimulation with 50 μg/ml anti-IgG in the presence of (+) or absence (-)of 20 μg/ml vesnarinone, determined using in situ hybridization. Alsoshown, are cells subsequently resuspended in fresh medium with(post-treatment +) or without (post-treatment -) 20 μg/ml vesnarinone,and then stimulated with 50 μg/ml of anti-IgG. The results are presentedas percentages relative to the value in vesnarinone-untreated cells.

FIGS. 12A-12C represent Western blots of lysates from four differentBurkitt's lymphoma cell lines, i.e., Akata, P3HR1, Jijoye, and Raji,cultured in the absence (-) or presence (+) of 20 μg/ml vesnarinone for7 days. Two EBV latent gene products, EBNA2 (FIG. 12A) and LMP1 (FIG.12B), and one host gene product, bcl-2 (FIG. 12C), were evaluated.

FIGS. 13A-13B show thymidine kinase assays of extracts fromDMSO-released Akata cell stimulated without (-) or with (+) 50 μg/ml ofanti-IgG antibody in the presence (+) or absence (-) of 100 μg/mlvesnarinone for 13 hrs. Whole thymidine kinase activity was measured inthe absence of dTTP (FIG. 13A). For determining dTTP-resistant thymidinekinase activity, 100 μM dTTP was added to the kinase reaction mixture(FIG. 13B).

FIGS. 14A and 14B show inhibition of uptake of ³ H!thymidine, but notAZT, by vesnarinone in a dose-dependent manner in CHO cells (FIG. 14A)and in primary human T lymphocytes (FIG. 14B).

FIG. 15A shows that vesnarinone delays the accumulation ofphosphorylated ³ H!thymidine in cellular extracts of PHA-activated andlymphocytes after removal of chromosomal debris; and FIG. 15B shows thatvesnarinone enhances the phosphorylation of AZT in PHA-activated human Tlymphocytes.

FIG. 16A shows that thymidine is effectively phosphorylated up to thetriphosphate level in cells. FIG. 16B shows that AZT is phosphorylatedto AZT-MP, but poorly phosphorylated beyond this stage to thediphosphate form.

FIG. 17 shows that vesnarinone, at a concentration of 75 μM (30 μg/ml),increased intracellular AZT-MP, AZT-DP, and AZT-MP by 100%, 45% and 25%,respectively.

FIGS. 18A and 18B show that both vesnarinone (FIG. 18A) and dypridamole(FIG. 18B) inhibit the uptake of thymidine in a dose-dependent manner;and that dipyridamole is a more potent inhibitor of nucleoside transportthan vesnarinone; the IC₅₀ for thymidine uptake was <10 nM indipyridamole, and -25 μM in vesnarinone.

FIG. 19A shows that vesnarinone increases the concentration ofphosphorylated AZT in U937 cells in a dose-dependent manner FIG. 19Bshows that 0-100 nM dipyridamole, which inhibits thymidine transport toa similar extent as 0-250 μM vesnarinone, does not affect intracellularconcentrations of phosphorylated AZT.

FIG. 20A shows the production of HIV-1 p24 antigen by AZT-sensitive HIVstrain 18a-infected PBMC in the presence or absence of vesnarinoneand/or AZT at various concentrations. FIG. 20B shows the production ofHIV-1 p24 antigen by AZT-resistant HIV strain 18c-infected PBMC in thepresence or absence of vesnarinone and/or AZT at various concentrations.

DETAILED DESCRIPTION OF THE INVENTION

In general formula (1), the benzoyl group which may have lower alkoxygroups and substituents on the phenyl ring, includes benzoyl groupshaving 1 to 3 straight-chain or branched C₁₋₆ alkoxy groups substitutingthe phenyl ring, such as benzoyl, 2-methoxybenzoyl, 3-methoxybenzoyl,4-methoxybenzoyl, 2-ethoxybenzoyl, 3-ethoxybenzoyl, 4-ethoxybenzoyl,4-isobutoxybenzoyl, 4-hexloxybenzoyl, 3,4-dimethoxybenzoyl,3,4-diethoxybenzoyl, 3,4,5-trimethoxybenzoyl, 2,5-dimethoxybenzoyl, andso

Of the active ingredient compound (1) according to the invention,3,4-dihydro-6- 4-(3,4-imethoxybenzoyl)-1-piperazinyl!-2(1H)-quinoline,i.e., vesnarinone, is most preferable.

The above carbostyrils will readily form a salt with a conventionalacid. As such acids, there may be mentioned inorganic acids, such assulfuric acid, nitric acid, hydrochloric acid and hydrobromic acid; andorganic acids, such as acetic acid, p-toluenesulfonic acid,ethanesulfonic acid, oxalic acid, maleic acid, fumaric acid, citricacid, succinic acid and benzoic acid. These salts can also be used asthe active ingredient in the present invention, just as can the freecompound of general formula (1).

The compounds of general formula (1) and salts thereof, can be generallyformulated into the per se conventional pharmaceutical preparations.Such preparations are prepared using conventional fillers, extenders,binding agents, moistening agents, disintegrating agents, surfactants,lubricants, and the like diluents or excipient. These pharmaceuticalpreparations may have various dosage forms selected according to thepurposes of therapy, and typical examples thereof are tablets, pills,powders, solutions, suspensions, emulsions, granules, capsules,suppositories, injections (solutions, suspensions, etc.), and ophthalmicsolutions.

For the manufacture of tablets, a wide variety of carriers so farwell-known in this field can be used. Thus, use can be made of, forexample, vehicles or excipient, such as lactose, sucrose, sodiumchloride, glucose, urea, starch, calcium carbonate, kaolin, crystallinecellulose and silicic acid; binding agents, such as water, ethanol,propanol, simple syrup, glucose solution, starch solution, gelatinsolution, carboxymethylcellulose, shellac, methylcellulose, potassiumphosphate and polyvinylpyrrolidone; disintegrating agents, such as drystarch, sodium alginate, powdered agar, powdered laminaran, sodiumhydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fattyacid esters, sodium lauryl sulfate, stearic acid monoglyceride, starchand lactose; disintegration inhibitors, such as sucrose, stearin, cacaobutter and hydrogenated oils; absorption promoters, such as quaternaryammonium bases and sodium lauryl sulfate; wetting agents or humectants,such as glycerol and starch; adsorbents, such as starch, lactose,kaolin, bentonite and colloidal silica; and lubricants, such as refinedtalc, stearic acid salts, powdered boric acid and polyethylene glycol.When necessary, the tablets may further be provided with a conventionalcoating to give, for example, sugar-coated tablets, gelatin-coatedtablets, enteric-coated tablets, film-coated tablets, or double-coatedor multilayer tablets.

For the manufacture of pills, a wide variety of carriers well-known inthe art can be used. Examples are vehicles or excipients, such asglucose, lactose, starch, cacao butter, hardened vegetable oils, kaolinand talc; binding agents, such as powdered gumarabic, powderedtragacanth gum, gelatin and ethanol; and disintegrating agents, such aslaminaran and agar.

For the manufacture of suppositories, a wide variety of known carrierscan be used. As examples, there may be mentioned polyethylene glycol,cacao butter, higher alcohols, higher alcohol esters, gelatin andsemisynthetic glycerides.

In preparing injections, the solutions or suspensions are preferablysterilized and are preferably isotonic with blood and, for preparingsuch dosage forms, all of the diluents in conventional use in the fieldcan be employed. Thus, for example, water, ethyl alcohol, propyleneglycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcoholand polyoxyethylene sorbitan fatty acid esters may be mentioned. In thiscase, the pharmaceutical preparations may contain sodium chloride,glucose or glycerol in an amount sufficient to give isotonic solutions.It is possible to add conventional solubilizing agents, buffers,soothing agents or local anesthetics, etc.

Furthermore, when necessary, the pharmaceutical preparations may containcoloring matters, preservatives, perfumes, flavoring agents, sweeteningagents and the like, as well as other drugs.

The proportion of the active ingredient compound in these pharmaceuticalpreparations for use in the present invention is not critical, and maysuitably be selected over a wide range. Generally, however, theproportion is recommendably selected within the range of about 1.0 toabout 70% by weight, preferably about 1.0 to about 30% by weight.

The route of administration of the pharmaceutical preparations of thepresent invention is not critical, either, but is selected according tothe dosage form, the patient's age, sex and other factors, and theseverity of the disease to be treated. Thus, for instance, when they areprovided in the form of tablets, pills, solutions, suspensions,emulsions, granules or capsules, the preparations are administeredorally. Injectable solutions are administered intravenously, eitheralone or in admixture with conventional fluids for parental infusioncontaining glucose, amino acids and so on. Where necessary, thesesolutions may also be administered as is by the intramuscular,intradermal, subcutaneous or intraperitoneal route. Suppositories areadministered rectally, ophthalmic solutions are drop lotions for theeyes.

While the dosage of the above pharmaceutical preparations is dependenton the method of administration, the patient's age, sex and otherbackground factors, severity of the disease and so on, it is generallyrecommended to administer about 0.5 to 30 mg, as the active ingredient,viz. compound (1), per kilogram body weight per day. The amount of theactive ingredient to be contained in each dosage unit is about 10 to1000 mg.

DOSAGE FORM EXAMPLE 1

    ______________________________________                                        3,4-dihydro-6- 4-(3,4-dimethoxybenzoyl)-1-                                                             150     g                                            piperazinyl!-2(1H)-quinoline                                                  Avicel                   40      g                                            (trademark, Asahi Chemical Industry, Co., Ltd.)                               Corn starch              30      g                                            Magnesium stearate       2       g                                            Hydroxypropylmethylcellulose                                                                           10      g                                            Polyethylene glycol 6000 3       g                                            Castor oil               40      g                                            Methanol                 40      g                                            ______________________________________                                    

The above active ingredient, Avicel, corn starch and magnesium stearateare mixed and ground together, and the resulting mixture iscompression-molded with a dragee R10 mm punch. The tablets thus obtainedare coated with a film coating composition consisting of hydroxypropylmethylcellulose, polyethylene glycol 6000, castor oil and methanol togive film-coated tablets.

DOSAGE FORM EXAMPLE 2

    ______________________________________                                        3,4-dihydro-6- 4-(3,4-dimethoxybenzoyl)-1-                                                             150     g                                            piperazinyl!-2(1H)-quinoline                                                  Citric acid              1       g                                            Lactose                  33.5    g                                            Dicalcium phosphate      70      g                                            Pluronic F-68            30      g                                            Sodium lauryl sulfate    15      g                                            Polyvinylpyrrolidone     15      g                                            Polyethylene glycol (Carbowax 1500)                                                                    4.5     g                                            Polyethylene glycol (Carbowax 6000)                                                                    45      g                                            Corn starch              30      g                                            Dry sodium lauryl sulfate                                                                              3       g                                            Dry magnesium stearate   3       g                                            Ethanol                  q.s.                                                 ______________________________________                                    

The above active ingredient, citric acid, lactose, dicalcium phosphate,pluronic F-68 and sodium lauryl sulfate are admixed.

After size selection using a No. 60 screen, the mixture is granulated bythe wet process using an alcoholic solution containingpolyvinylpyrrolidone, Carbowax 1500 and Carbowax 6000. When necessary,alcohol is added to make the powder into a paste-like mass. Then, cornstarch is added, and the blending is continued until uniform granulesare formed. The mixture is then passed through a No. 10 screen, placedin a tray and dried in an oven maintained at 100° C. for 12 to 14 hrs.The dried granules are sieved through a No. 16 screen, then dry sodiumlauryl sulfate and dry magnesium stearate are added and, after blending,the mixture is compressed to a desired size and shape using a tabletingmachine.

The above cores are treated with a varnish and dusted with talc forpreventing absorption of moisture, and then provided with an undercoatlayer. Varnish coating is repeated as many times as sufficient forinternal use. The tablets are rendered completely round and smooth byapplication of a further undercoat layer and a smooth coating. Colorcoating is conducted until a desired coloring is obtained. After drying,the coated tablets are polished to give uniformly polished tablets.

The DNA viruses whose replication can be inhibited in the presentinvention include: herpes simplex virus type 1 and 2, human herpes virustype 6, herpes zoster virus, human cytomegalovirus and EBV. EBV is thepreferred DNA virus whose replication can be inhibited in the presentinvention.

Thus, the above carbostyrils are useful in the treatment of disordersassociated with chronic EBV infection, e.g., chronic fatigue syndromeand chronic infectious mononucleosis; EBV-associated lymphoma, e.g.,Burkitt's lymphoma, B cell lymphoma, T cell lymphoma, nasopharyngealcarcinoma and Hodgkin's disease; or EBV-induced lymphoproliferative; orEBV-associated autoimmune disease, e.g., Sj0gren syndrome.

In addition, in the methods of inhibiting DNA virus replication, andparticularly treating EBV infection, the carbostyrils of the presentinvention can be used in combination with other known anti-DNA viruscompounds, particularly, other known commercially available anti-EBVcompounds, such as Acyclovir (9- (2-hydroxyethoxy)methyl!guanine)(Burroughs Wellcome); and Ganciclovir sodium9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG) (Syntex). In thismanner, the dosage conventionally used for the known anti-DNA viruscompounds can be reduced. The particular dosage of anti-DNA viruscompounds.

The RNA viruses whose replication can be inhibited in the presentinvention include: human immune deficiency virus (HIV), adult T-cellleukemia virus, and human immune deficiency virus type II. HIV is thepreferred RNA virus whose replication can be inhibited in the presentinvention.

Thus, the above carbostyrils are useful in the treatment of disordersassociated with HIV infection, e.g., Kaposi's sarcoma, hemologicconsequences, and auto-immunity.

In addition, in the methods of inhibiting virus replication, andparticularly treating HIV infection, the carbostyrils of the presentinvention can be used in combination with other known anti-RNA viruscompounds, particularly, other known commercially available anti-HIVcompounds, such as AZT (3'azido-3'-deoxythymidine) (Burroughs Wellcome);ddC (2'3'-dideoxycytidine) (Hoffmann LaRoche), ddI (2'3'-dideoxyinosine)(B.M.-Squibb), d4T (2'3'-didehydro-2'3'-dideoxythymidine) (B.M.-Squibb),and 3TC (5'3'-dideoxythiacytidine) (Glaxo). In this manner, the dosageconventionally used for the known anti-RNA virus compounds can bereduced.

The particular nucleoside analogues whose phosphorylation is enhancedwhen co-administered with the carbostrylis of the present invention arenot limiting. Examples of such nucleoside analogues include theabove-listed anti-DNA virus compounds and anti-RNA virus compounds,particularly AZT.

The following examples are provided for illustrative purposes only, andare in no way intended to limit the scope of the present invention.

EXAMPLE 1 A. Vesnarinone Blocks Nucleoside Incorporation ImmediatelyAfter Addition of the Drug

Vesnarinone has been reported to have a mild cytostatic effect dependingon the cell type tested (Nakai et al, Jpn J. Cancer Res., Abstract,Proc. Jpn. Cancer Assoc., page 581 (1993)). Thus, in order toinvestigate the mechanism of action of vesnarinone, nucleoside ornucleobase transport into cells was measured as described by Aronow etal, J. Biol. Chem., 260:16274-16278 (1985).

More specifically, human primary T cells were separated from peripheralblood obtained by leukopheresis of healthy donors. Mononuclear cellsuspensions were prepared by Ficoll-Hypaque gradient centrifugation, andT cells were obtained by E-rosette enrichment as described by Kumagai etal, J. Cell. Physiol., 137:329 (1988). The resulting T cells werestimulated for 48 hrs with 10 μg/ml phytohemagglutinin (PHA), in theabsence of any drug (referred to as "PHA") and in the presence of HEPESbuffer (referred to as "vehicle") as controls, or in the presence of0-250 μM vesnarinone, prepared as described below.

10 mg of vesnarinone was dissolved in 0.5 ml of 2.0N HCl. The solutionwas added to 9.0 ml of RPMI 1640 medium supplemented with 20 mM HEPESbuffer (pH 7.55), and neutralized to pH 7.0 by addition of 2.0N NaOH.The resulting neutralized vesnarinone solution containing 1.0 mg/ml ofvesnarinone was immediately filtered using a 0.45 μm Millipore filter,and added to the cell cultures (1 μg/ml≈2.5 μM of vesnarinone).

Next, the cells were resuspended in RPMI 1640 medium (GIBCO, GrandIsland, N.Y.) supplemented with 10% (v/v) heat-inactivated fetal bovineserum (FBS, HyClone, Logan, Utah), 2.0 mM L-glutamine (GIBCO), 100 U/mlpenicillin and 100 μg/ml streptomycin (GIBCO), and containing 20 mMHEPES (pH 7.4), and incubated for 20 min at 37° C.

Then, 1.0 μCi of ³ H!thymidine (6.7 Ci/mmole, ICN), was added to 2.0×10⁶cells (100 μl), and the cells were pulse-labeled for 6 hrs. Next, themixture was immediately layered on silicone oil/paraffin oil (94:6) inmicrofuge tubes, and the cells were separated from free ³ H!substrate inthe water phase by spinning for 1 min at 14,000 rpm.

The water and oil phases were discarded, and the radioactivity in thecell pellets was measured using liquid scintillation counting. Theresults are shown in FIG. 1A.

As shown in FIG. 1A, vesnarinone inhibits ³ H!thymidine incorporationinto DNA in a dose-dependent manner.

Next, 15 μM aphidicolin and 100 μM deferoxamine were obtained, andutilized as described by Terada et al, J. Immunol., 147:698-704 (1991).The results are shown in FIG. 1B.

As shown in FIG. 1B, addition of aphidicolin, which inhibits DNApolymerase α/δ, or deferoxamine, which inhibits ribonucleobasereductase, also inhibited ³ H!thymidine incorporation.

Next, the effects of the above-described drugs on cell proliferationwere evaluated over a period of 120 hrs. More specifically, the aboveexperiment was repeated Using 250 μM vesnarinone, 15 nM aphidicolin or100 μM deferoxamine, and 10 μg/ml PHA. The results are shown in FIG. 1C.

As shown in FIG. 1C, a high dose of vesnarinone (250 μM) inhibited cellproliferation when compared to control cultures, but increases in cellnumber were nonetheless observed. In contrast, as shown in FIG. 1C, noincreases in cell number were observed in the presence of aphidicolin ordeferoxamine. Again all three drugs inhibited ³ H!thymidineincorporation to a similar degree at the doses indicated in FIG. 1B.

Next, cellular DNA content was evaluated over the 0-120 hrs treatmentperiod. More specifically, 10⁶ T cells treated in the same manner asdescribed above, were fixed with 70% (v/v) ethanol at 4° C. overnight,centrifuged and washed with phosphate buffered saline (PBS). The fixedcells were incubated with 0.5 ml of 0.25 mg/ml ribonuclease (Sigma, St.Louis, Mo.) in PBS at 37° C. for 10 min. The cell suspension was mixedwith 0.5 ml of a 50 μg/ml propidium iodide (Calbiochem, La Jolla,Calif.) solution in PBS, and after 60 min analyzed by flow cytometry(EPICS Profile, Coulter, Hialeah, Fla.), collecting red fluorescence(>600 nm) with 488 nm excitation. The results are shown in FIGS. 2A-2F.

As shown in FIGS. 2A-2F, consistent with the increases in cell number,monitoring of cellular DNA content revealed that a certain proportion ofthe cells entered both S and G2/M phases of the cell cycle in thepresence of vesnarinone within 48 hrs, but not in the presence ofaphidicolin or deferoxamine. ³ H!thymidine incorporation was inhibitedby vesnarinone to a similar degree at all time points throughout theexperiment (up to 120 hrs), and there was no recovery of ³ H!thymidineincorporation at later time points. Thus, these results indicate thatvesnarinone may inhibit ³ H!thymidine incorporation by a mechanism otherthan inhibition of DNA synthesis per se.

In order to further evaluate the interaction of vesnarinone on ³H!thymidine and ³ H!uridine incorporation, the effects of brief exposureof proliferating cells to vesnarinone were examined.

More specifically, 10⁶ T cells were stimulated with 10 μg/ml PHA for 48hrs in the presence of 250 μM vesnarinone or 15 nM aphidicolin or 100 μMdeferoxamine, and ³ H!thymidine or ³ H!uridine (35.1 Ci/mmole, ICN) wereadded for only 30 min, and cell pellets were obtained and evaluated asdescribed above. The results are shown in FIG. 3.

As shown in FIG. 3, vesnarinone and aphidicolin were able to inhibit ³H!thymidine incorporation under these conditions as well. In contrast,as shown in FIG. 3, deferoxamine only partially inhibited ³ H!thymidineincorporation, likely because increases in dNTP pools in proliferatingcells were sufficient to sustain DNA synthesis for a period of timewithout further supply of dNTP.

In addition, as shown in FIG. 3, vesnarinone rapidly inhibited ³H!uridine incorporation as well. This was also in contrast to the verysmall effect on cellular RNA content, measured by flow cytometryfollowing acridine orange staining of cells. Aphidicolin or deferoxaminedid not inhibit ² H!uridine incorporation.

Next, the PHA-stimulated T cells were labelled for 30 min with 5.0μCi/ml of an ³ H!amino acid mixture (1.0 mCi/ml, Amersham, ArlingtonHeights, Ill.), washed twice with ice-cold PBS, and then lysed in PBScontaining 1.0% (w/v) SDS, followed by addition of a mixture of 7.0%(w/v) trichloroacetic acid and 1.0% (w/v) pyrophosphate. Theprecipitates were loaded on GF/A filters, and washed extensively with amixture of 7.0% (w/v) trichloroacetic acid and 1.0% (w/v) pyrophosphate.Radioactivity was measured by scintillation counting. The results arealso shown in FIG. 3.

As shown in FIG. 3, vesnarinone did not inhibit ³ H!amino acidincorporation.

Thus, the profound and rapid inhibition of both ³ H!thymidine and ³H!uridine incorporation by vesnarinone is not likely explained by adirect effect on DNA and RNA synthesis. The discrepancy suggests thatvesnarinone may interfere with nucleoside incorporation into cells.

B. Vesnarinone Inhibits Nucleoside and Nucleobase Transport

Nucleosides permeate the plasma membrane of cells, and are utilized as asource for the Salvage pathway of nucleobase synthesis (Cory, In:Biochemistry with Clinical Correlations, Chapter 13, pages 529-571(1992); and Rodwell, In: Biochemistry, Chapter 36, pages 363-377(1993)). The transport of nucleosides into mammalian cells occurs by afacilitated diffusion mechanism which appears to be mediated by a singleor multiple carriers of variable specificity (Aronow et al, J. Biol.Chem., 261:14467-14473 (1986)). This carrier model is based on kineticevidence indicating competitive inhibition among nucleosides fortransport and on the results in mutant cells which are geneticallydeficient in nucleoside transport capability (Aronow et al, J. Biol.Chem., 261:14467-14473 (1986); Cohen et al, J. Biol. Chem., 254:112-116(1979); and Ullman et al, Mol. Cell. Biol., 3:1187-1196 (1983)).

The study of nucleoside transport in mammalian cells has been greatlyenhanced by the existence of specific high affinity inhibitors of thenucleoside transporter in mammalian cells, including4-nitrobenzyl-6-thioinosine (NBMPR), dipyridamole and dilazep (Aronow etal, J. Biol. Chem., 260:6226-6233 (1985); Scholtissek et al, Biochem.Biophys. Acta, 158:435-447 (1968); Plagemann et al, J. Membr. Biol.,81:255-262 (1984); Berlin et al, Int. Rev. Cytol., 42:287-336 (1975);and Fujita et al, Br. J. Pharmacol., 68:343-349 (1980)). The entry ofnucleobases, another physiological source for salvage nucleobasesynthesis, is less well defined.

Nucleosides and nucleobases mutually interfere with the translocation ofeach other in some animal cells, suggesting a role for the nucleosidetransporter in nucleobase transport (Aronow et al, J. Biol. Chem.,261:2014-2019 (1986)). On the other hand, the presence of independentnucleobase transport is suggested through studies using mutant celllines (Aronow et al, J. Biol. Chem., 261:2014-2019 (1986)). Thus, thepossibility that vesnarinone inhibits nucleoside transport wasevaluated, by looking at rapid transport of radiolabelled nucleosidesand nucleobases into T cells.

More specifically, 10⁶ T cells stimulated for 48 hrs with 10 μg/ml PHAwere treated with 250 μM vesnarinone, 100 μM deferoxamine, 15 nMaphidicolin or 1.0 μM dipyridamole, and pulse-labelled for 1 min with ³H!thymidine, ³ H!adenosine (23 Ci/mmole, Amersham), ³ H!adenine (26Ci/mmole, Amersham) or ³ H!uracil (53 Ci/mmole, Amersham), and cellpellets were obtained and evaluated as described above. The results areshown in FIG. 4.

As shown in FIG. 4, vesnarinone inhibited transportation of ³H!thymidine, ³ H!adenosine, ³ H!adenine, and also to a lesser extent, ³H!uracil. In contrast, deferoxamine or aphidicolin had no effect on thetransport of these nucleosides or nucleobases, despite using the sameconcentration of these drugs which inhibited ³ H!thymidine incorporationinto DNA. In addition, dipyridamole, a specific high affinity inhibitorof a nucleoside transporter (Scholtissek et al, Biochem. Biophys. Acta,158:435-447 (1968); and Plagemann et al, J. Membr. Biol., 81:255-262(1984)), suppressed nucleoside transport, but not nucleobase transport.

Next, dose-dependent responses to vesnarinone on nucleoside andnucleobase transport were examined using several different cell lines,i.e., human lymphoblastoid line, Raji, which was obtained from theAmerican Type Culture Collection (ATCC No. CCL-86), and the Chinesehamster ovary cell line, CHO, which was provided by Dr. G. L. Johnson(Denver, Colo.).

More specifically, 2.0×10⁶ Raji or CHO cells were treated with 0-750 μMvesnarinone or 0-100 μM dipyridamole, and pulse-labelled for 1 min with³ H!adenine or ³ H!adenosine, and the cell pellets obtained andevaluated as described above. The results are shown in FIGS. 5A-5D.

As shown in FIG. 5A, in the presence of vesnarinone, Raji cells had anincreased ability to transport the purine nucleobase, ³ H!adenine in adose-dependent manner, while, as shown in FIG. 5C, CHO cells, in thepresence of vesnarinone, had a lesser ability to transport thisnucleobase. In contrast, as shown in FIGS. 5A and 5C, in the presence ofvesnarinone both cells showed a similar ability to transport the purinenucleoside, adenosine in a dose-dependent manner. Such variability isnot surprising since variability, especially for nucleobase transport,has been reported previously (Aronow et al, Mol. Cell Biol., 6:2957-2962(1986)).

It should be noted that the inhibitory effects on nucleoside andnucleobase transport in vitro were observed within the therapeutic rangeof vesnarinone in vivo (2-80 μM) (shown as a bar in FIGS. 5A and 5C).

In contrast, as shown in FIGS. 5B and 5D, dipyridamole inhibitedadenosine transport in a selective fashion with no effect on adeninetransport in its therapeutic range (10-100 nM) (also shown as a bar inFIGS. 5B and 5D). Only high concentrations of dipyridamole (10-100 μM)inhibited adenine transport, as described by Scholtissek et al, Biochem.Biophys. Acta, 158:435-447 (1968); and Plagemann et al, J. Membr. Biol.,81:255-262 (1984).

Transport of other nucleosides, including thymidine and uridine, wasaffected by vesnarinone in a manner similar to adenosine, whiletransport of uracil (a pyrimidine nucleobase) was inhibited likeadenine, but to a lesser extent.

These data indicate that vesnarinone inhibits nucleoside and nucleobasetransport, but via a different mechanism from that of dipyridamole.Furthermore, FIGS. 5A and 5C illustrate that the degree of inhibition ofnucleoside transport by vesnarinone was similar to that of nucleosideincorporation demonstrated in FIG. 1A. These results indicate that mostof the inhibition of ³ H!nucleoside incorporation by vesnarinone is dueto inhibition of nucleoside transport by vesnarinone.

Nucleoside transport inhibitors, such as dipyridamole or NBMPR, areknown to rescue cell proliferation from the cytotoxic effects of5-fluorouridine (5-FUd) or high dose thymidine, by inhibiting transportof this cytotoxic nucleoside analog or thymidine (Aronow et al, Mol.Cell Biol., 6:2957-2962 (1986)). Thus, vesnarinone was tested todetermine whether it has a similar effect using CHO cells.

More specifically, 1×10⁴ CHO cells/ml were incubated with 50 nM 5-FUd inthe presence of 0-250 μM vesnarinone or 1.0-10 μM dipyridamole, and cellnumbers were counted after 120 hrs of incubation. The results are shownin FIGS. 6A-6B.

As shown in FIG. 6B, dipyridamole significantly reversed the cytostaticeffects of 5-FUd. Also as shown in FIG. 6A, vesnarinone demonstrated asimilar ability to rescue the cells in a dose-dependent manner. It wasdifficult to demonstrate a similar effect when higher (≧100 nM) or lower(≦10 nM) doses of 5-FUd were used, likely due to the anti-proliferativeeffects of vesnarinone itself. These data provide additional supportthat vesnarinone acts as an inhibitor of nucleoside transport.

In this Example it was demonstrated that vesnarinone inhibits nucleosideand nucleobase transport, while other high affinity inhibitors ofnucleoside transport, such as dipyridamole, have a markedly reducedability to inhibit nucleobase transport. Although much higherconcentrations of vesnarinone are required, with vesnarinone, it isnotable that the inhibitory effects on nucleoside and nucleobasetransport in vitro were observed within the therapeutic range ofvesnarinone in vivo (2-80 μM), implying that these effects in vitro maybe related to some clinical effects of vesnarinone.

Inhibition of nucleoside and nucleobase uptake into cells by vesnarinonecould result in the inhibition of cell proliferation, especially wherenucleobase synthesis is more dependent on the salvage pathway. Celltypes, such as polymorphonuclear leukocytes, certain brain cells,intestinal mucosal cells or erythrocytes have little or no de novosynthesis capability for nucleobases, whereas other cells such ashepatocytes have an active de novo synthesis system. Therefore, these invitro observations may delineate the cause of agranulocytosis, a majorside effect of vesnarinone observed in some patients (OPC-8212Multicenter Research Group, Cardiovasc. Drugs, Ther., 4:419-425 (1990);Feldman et al, Am. J. Cardiol., 68:1203-1210 (1991); Feldman et al, N.Engl. J. Med., 329:149-155 (1993); and Packer, N. Engl. J. Med.,329:201-202 (1993)).

The decrease in nucleoside and nucleobase transport by vesnarinone mayalso play a role in the inhibitory effect on HIV-1 replication(Meyerhans et al, J. Virol., 68:535-540 (1994); O'Brien et al, J.Virol., 68:1258-1263 (1994); and Gao et al, Proc. Natl. Acad. Sci. USA,90:8925-8928 (1993)). Recently, triggering decreases or an imbalance innucleobase pools has been reported to reduce the rate of reversetranscription of HIV-1 in cells or to induce aborted viral replication(Ullman et al, Mol. Cell. Biol., 3:1187-1196 (1983)); Aronow et al, J.Biol. Chem., 260:6226-6233 (1985); and Scholtissek et al, Biochem.Biophys. Acta, 158:435-447 (1968)).

Finally, inhibition of adenosine transport may provide the link withanother novel aspect in the action of vesnarinone. Dipyridamole isproposed to cause a localized increase in adenosine concentrationthrough its inhibition of adenosine transport into cells (Plagemann etal, Biochem. Biophys. Acta, 947:405-443 (1988)). Adenosine is known toinduce an increase in cAMP (Fox et al, Ann. Rev. Biochem., 47:655-686(1978)), dilation of coronary arteries (Fox et al, Ann. Rev. Biochem.,47:655-686 (1978)), an increase in cerebral blood flow (Heistad et al,Am. J. Physiol., 240:775-780 (1981)), a decrease in TNF-α production(Parmely et al, J. Immunol., 151:389-396 (1993)), and a decrease inplatelet aggregation (Dawicki et al, Biochem. Pharmacol., 34:3965-3972(1985)), through its binding to specific adenosine receptors on cellsurface membranes. Vesnarinone might similarly increase bloodconcentrations of adenosine by inhibiting adenosine transport, thusexplaining some of the therapeutic benefit of vesnarinone in heartdisease (Feldman et al, N. Engl. J. Med., 329:149-155 (1993); andPacker, N. Engl. J. Med., 329:201-202 (1993)) or in the reduction ofTFN-α production (Maruyama et al, Biochem. Biophys. Res. Comm.,195:1264-1271 (1993); and Matsumori et al, Circul., 89:955-958 (1994)).

EXAMPLE 2 A. Effect of Vesnarinone on Expression of ZEBRA and onProductive Replication of EBV

The human Burkitt's lymphoma cell line, Akata, provided by Dr. KenzoTakada (Takada, Int. J. Cancer, 33:27-32 (1984); and Takada et al, VirusGenes, 5:147-156 (1991)), was cultured at 37° C. in RPMI 1640 mediumsupplemented with 15% (v/v) heat-inactivated FCS, and 20 μg/mlgentamicin.

The cell progression of Akata cells was arrested by culturing of thecells for 96 hrs in the presence of 1.5% (y/v) DMSO (Sigma, St. Louis,Mo.) (Sawai et al, Exp. Cell Res., 187:4-10 (1990); and Takase et al,Cell Growth Differ., 3:515-521 (1992)). Then, the DMSO-arrested Akatacells were washed twice with PBS, and transferred, at a density of 10⁶/ml, to RPMI 1640 medium containing vesnarinone at differentconcentrations ranging from 0-100 μg/ml, prepared as described below.

10 mg vesnarinone was dissolved in 0.5 ml of 2.0N HCl and then dilutedwith 5.0 ml of H₂ O and 4.0 ml of RPMI 1640 medium. After neutralizationwith 2.0N NaOH (about 0.5 ml), the vesnarinone solution (1.0 mg/ml) wasimmediately filtered through a 0.45 μm Millipore filter, and diluted tothe desired concentration in culture medium.

1 hr after transfer (release from DMSO), a goat F(ab')₂ fragment ofanti-human IgG antibody (Organon Teknika, Durham, N.C.) was added at aconcentration of 50 μg/ml to induce productive EBV replication (Takagiet al, Virology, 185:309-315 (1991)).

3 hrs and 20 hrs after treatment with the anti-IgG, the cells wereharvested for flow cytometric detection of the BamHI Z replicationactivator (ZEBRA), and for in situ detection of EBV DNA, respectively.

For flow cytometric detection of ZEBRA, after a 60 min fixation with 70%(v/v) ethanol at 4° C., 10⁶ cells were centrifuged, and washed with PBS.After a 5 min incubation in 250 μl of PBS containing 0.5% (v/v)Tween-20, 2.0% (w/v) BSA and 1.5% (w/v) human γ-globulin (Sigma) at roomtemperature, 10 μl of the hybridoma supernatant of mouse monoclonalanti-EB1/ZEBRA (Z-125, provided by Dr. Evelyn Manet) (Mikaelian et al,J. Virol, 67:734-742 (1993)) was added, and the mixture was incubated at4° C. for 120 min. The cells were then washed with PBS, and furthertreated with 5.0 μl of a FITC-conjugated F(ab')₂ fragment of affinitypurified sheep anti-mouse IgG (F/P molar ratio=3.2, Sigma) at 4° C. for60 min. The cells were washed with PBS again, and analyzed by flowcytometry (Epics Profile, Coulter), monitoring fluorescence (520-540 nm)in a logarithmic scale with 488 nm excitation. The positivity for ZEBRAwas calculated by a non-linear least squares method (Takase et al, J.Immunol. Methods, 118:129-138 (1989)).

For detection of EBV DNA in cells, an in situ hybridization techniqueusing the kit from ENZO Diagnostics (Syosset, N.Y.) was employed (Takagiet al, Virol., 185:309-315 (1991)). The hybridization procedure wasperformed basically according to the instructions of the manufacturer.

More specifically, cells coated on a glass slide were fixed in acetoneat room temperature for 10 min. Target and probe DNA (biotinylated probeof EBV BamHI Z fragment) were denatured at 94° C. for 4 min, andhybridized for 20 min at 37° C. For detection of the specificallyhybridized DNA, the slide was treated with an avidin-biotinylatedhorseradish peroxidase complex, and developed by3-amino-9-ethylcarbazole and hydrogen peroxide. Positivity for EBV DNAwas determined by the presence of red-colored grains and at least 1000cells were examined under light microscopy. Cells in which productiveEBV replication is ongoing are plainly distinguished by the strongstaining (Takagi et al, Virol., 185:309-315 (1991)). It should be notedthat both positivity for EBV DNA in in situ hybridization and multiplelinear forms of EBV DNA demonstrated in Southern blotting (BamHIdigestion and NJhet DNA probed) (Raab-Traub et al, Cell, 47:883-889(1986)) are dramatically reduced by acyclovir pretreatment in anti-IgGtreated Akata cells. The results are shown in FIG. 7.

As shown in FIG. 7, the population of EBV producing cells, as measuredby EBV DNA, gradually decreased in the presence of vesnarinone in adose-dependent manner. On the other hand, the degree of positivity forZEBRA was not decreased by vesnarinone at concentrations less than orequal to 10 μg/ml. A shift in the curve was observed at concentrationsgreater than 30 μg/ml. Thus, susceptibilities to vesnarinone ofexpression of an early gene of the productive cycle, i.e., ZEBRA, andproductive replication of EBV genome could be distinguished.

B. Effects of Vesnarinone on Expression of Early Antigens

DMSO-arrested Akata cells were washed, and transferred to culture mediumcontaining 30 μg/ml vesnarinone. 1 hr after release and transfer, theabove-described anti-IgG antibody was added at a concentration of 50μg/ml. 3 hrs and 20 hrs after treatment with the anti-IgG, the cellswere harvested for the detection of EBV early antigens ZEBRA (BZLF1product) by Western immunoblot.

More specifically, cells were washed with PBS, and then lysed at 4° C.in a buffer comprising 25 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5% (w/v)sodium deoxycholate, 2.0% (v/v) Nonidet P-40, 0.2% (w/v) SDS, 1.0 mMphenyl-methane sulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 10 μMleupeptin, and 100 μM sodium orthovanadate. After centrifugation,lysates were prepared for electrophoresis as described by Laemmli,Nature, 227:680-685 (1970). An amount of lysate equivalent to 4.0×10⁵cells was used for each immunoblot analysis.

After electrophoretic transfer of proteins to nitrocellulose filters andblocking the filters with 20 mM Tris-HCl (pH 8.0), 125 mM NaCl, 0.1%(v/v) Tween-20, 2.0% (w/v) BSA, and 0.1% (w/v) sodium azide, the filterswere incubated with the appropriate primary antibody. For detection ofZEBRA (EB1, BZLF1 gene product), the antibody used was mouse monoclonalanti-EB1 (Z-125); for the detection of R (BRLF1 product), the antibodyused was the mouse monoclonal anti-R (R5A9, provided by Dr. EvelynManet); for the detection of EA-R (BHRF1 product), monoclonal anti-EA-R(5B11, provided by Dr. Elliott Kieff) (Pearson et al, Virol.,160:151-161 (1987); and Henderson et al, Proc. Natl. Acad. Sci. USA,90:8479-8483 (1993)), and for the detection of EA-D (BMRF1 product), theantibody used with the mouse monoclonal anti-EA-D (NEA-9240, DuPont,Boston, Mass. (Pearson et al, J. Virol., 47:193-201 (1983); and Kiehl etal, Virol., 184:330-340 (1991))).

Specific reactive bands were detected using anti-mouse secondaryantibodies conjugated to alkaline phosphatase (Promega, Madison, Wis.)or horseradish peroxidase (Amersham) or ECL chemiluminescence(Amersham). That is, for detection of ZEBRA and R, alkaline phosphatasewas used. For detection of EA-R and EA-D, horseradish peroxidase and theECL chemiluminescence method was used (Amersham). Development was by theconventional methods, employing the colorogenic substratesbromochloroindolyl phosphate and nitroblue tetrazolium for alkalinephosphatase. The expression of ZEBRA reached a plateau level 3 hrs afterstimulation and productive replication of EBV ceased 12 hrs afterstimulation in this system. The results are shown in FIGS. 8A-8D.

As shown in FIG. 8A, the expression of ZEBRA was suppressed 3 hrs afterstimulation with anti-IgG to a limited extent in the presence ofvesnarinone, comparable to the data presented in FIG. 7. As shown inFIG. 8B, another immediate early gene product, R was expressed virtuallyat the same level in the presence or absence of vesnarinone, 3 hrs and20 hrs after stimulation. As shown in FIG. 8C, the bcl-2 homologousearly gene product, EA-R was also expressed to the same degree 3 hrs and20 hrs after stimulation. In contrast, as shown in FIG. 8D, expressionof an EBV DNA polymerase cofactor, EA-D, which is expressed at a latertime point than the other three early genes, was dramatically suppressedin the presence of vesnarinone, 20 hrs after stimulation.

C. Cellular DNA Content of Anti-IgG Treated DMSO-Released Akata Cells inthe Presence of Vesnarinone

During the productive phase of EBV replication, a broadening of the 2Cpeak of DNA has been noted. This phenomenon presumably contributed tothe denaturation or disruption of chromosomal DNA as a part of the lyticprocess. In some cases, the extent of the change in the 2C peakcorrelated with the rate of EBV production. Thus, the 2C peak inanti-IgG treated DMSO-released Akata cells in the presence ofvesnarinone was evaluated.

More specifically, DMSO-released Akata cells stimulated with 50 μg/ml ofanti-IgG for 20 hrs in the presence of vesnarinone at concentrationsranging from 0-100 μg/ml as described above, were fixed overnight with70% (v/v) ethanol at 4° C., and 10⁶ cells were centrifuged, and washedwith PBS. Next, the fixed cells were incubated with 0.5 ml of 0.25 mg/mlribonuclease (Sigma) in PBS at 37° C. for 20 min. Then, the cellsuspension was mixed with 0.5 ml of propidium iodide (Calbiodiem, LaJolla, Calif.) solution (50 μg/ml in PBS), and after 60 min analyzed byflow cytometry (EPICS Profile, Coulter, Hialeah, Fla.), monitoringfluorescence (>600 nm) in a linear scale with 488 nm excitation (Takaseet al, Cell Growth Differ., 3:515-521 (1992)). The standard deviation ofthe G1 peak of the. DNA histogram was calculated by a non-linear leastsquares method (Takase et al, J. Immunol. Methods, 118:129-138 (1989)).The results are shown in FIGS. 9A-9F.

As shown in FIGS. 9A-9F, the distinctly broadened 2C peak, compared tothe sharp 2C peak observed in control samples without anti-IgGtreatment, were observed in samples treated with anti-IgG in thepresence or absence of vesnarinone. These observations indicate thatthis characteristic of the lytic process observed in anti-IgG treatedAkata cells was not prevented by vesnarinone, despite the reduction inEBV DNA replication.

D. dNTP Pools in Cells Treated with Vesnarinone

The profiles of four dNTPs in Akata cells were measured under severalconditions, in logarithmically growing cells, in DMSO-arrested cells, incells 13 hrs and 21 hrs after release from DMSO in the absence ofvesnarinone, and in cells 13 hrs and 21 hrs after release from DMSO inthe presence of 100 μg/ml vesnarinone.

More specifically, 10⁷ cells were washed with PBS, and vigorouslysuspended in 300 μl of cold 60% (v/v) ethanol (Tyrsted, Exp. Cell Res.,91:429-440 (1975)). After a 10 min incubation on ice, the supernatantcontaining dNTPs was removed, frozen and evaporated with a rotaryevaporator. The residue was dissolved in 100 μl of H₂ O, and a 10 μlaliquot of this solution was used for the measurement of dNTP pool.

In order to quantitate the amount of each dNTP, an enzymatic methodusing DNA polymerase I and calf thymus DNA as a substrate was employed(Williams et al, J. Biochem. Biophys. Methods, 1:153-162 (1979)).

More specifically, 10 μl of the sample or a dNTP standard solution wasmixed with 40 μl of the reaction buffer comprising 62.5 mMTris-HCl (pH7.8), 0.625 mM DTT, 0.125 mg/ml calf thymus DNA (Sigma), 0.625 mg/mlBSA, 10 mM MgCl₂, 6.25 μM three dNTPs, and 5.0 μCi/ml ³ H!dATP (ICN,Costa Mesa, Calif.), for measurement of dTTP, dCTP, and dGTP, or ³H!dTTP (ICN), for measurement of dATP. The reaction solution was thenmixed with 1.0 unit of E. coli DNA polymerase I (Boehringer Mannheim),and incubated for 40 min at 37° C. After completion of the reaction, themixture was applied to Whatman DE-81 paper filters and radioactivity wasdetermined using a liquid scintillation counter after three washes with0.35M Na₂ HPO₄ (pH 7.5). Amounts of dNTP in the samples were calculatedaccording to the calibration curves estimated by a set of standard dNTPsamples. Vesnarinone at concentrations up to 100 μg/ml has no effect onthe measurement of dNTP contents in this in vitro DNA polymerase assay.The results are shown in FIGS. 10A-10D.

As shown in FIGS. 10A-10D, the cellular content of all dNTPs wereuniformly reduced following treatment with DMSO. Both dATP and dGTPdisplayed progressive reductions 13 hrs after release in the presence ofvesnarinone without anti-IgG stimulation, despite the fact that thecellular contents were fairly well sustained at a relatively higherlevel or increased in the absence of vesnarinone. The cellular contentof dTTP was demonstrated to be the smallest storage pool among the fourdNTPs throughout all of the conditions. The reduced level of dTTP inDMSO-arrested cells was nearly restored to those in growing cells by 21hrs after release. On the other hand, in cells released from DMSO in thepresence of vesnarinone, the levels of dTTP remained quite low. Thelevels of dCTP remained low in the presence or absence of vesnarinone incells after release.

In parallel, to measurement of the content of dNTPs in the untreatedAkata cells after release, the dNTP pool in anti-IgG treated Akata cellswas also measured in the same manner. The results are shown in FIGS.10A-10D.

As shown in FIGS. 10A-10D, the level of dTTP at 12 hrs after treatmentwith anti-IgG (equivalently 13 hrs after release) was markedly elevated.This level of dTTP remained high 20 hrs after treatment, and waspartially suppressed in the presence of vesnarinone. As for the threeother dNTPs, such an increase in content as observed in dTTP was notdemonstrated. The effects of vesnarinone on dNTP in anti-IgG stimulatedcells was smaller than that in unstimulated cells except for dTTP.

E. Effects of Vesnarinone on Productive Replication of EBV in GrowingCells

Akata cells were maintained in growth phase for 7 days in culture mediumwith or without 20 μg/ml vesnarinone (pre-treatment). The cells werespun down, and then transferred to fresh culture medium with or without20 μg/ml vesnarinone (post-treatment). 1 hr later, anti-IgG was added tothe cultures at a final concentration of 50 μg/ml, and the cells wereharvested for in situ hybridization of EBV DNA 24 hrs later as describedabove. The results are shown in FIG. 11.

As shown in FIG. 11, treatment of the cells with vesnarinone for alonger period prior to (pre-treatment+post-treatment) or just prior to(solely post-treatment) addition of anti-IgG antibody reduced the levelof positivity for EBV DNA compared to untreated cells. Cells treatedwith vesnarinone only concurrently with anti-IgG treatment displayed amore profound reduction of EBV production. In addition, in the face ofpre-treatment alone, the degree of positivity for EBV DNA was higher.Thus, cell synchronously entering the lytic cycle may be moresusceptible to vesnarinone.

F. Effects of Vesnarinone on Expression of Molecules Related to Latentphase of EBV Infection

The effects of vesnarinone on the expression of molecules related to thelatent phase of EBV infection using four different EBV containing humanB cell lines, were examined, i.e., cells carrying two differentsubgroups of EBV, type A and type B (Adldinger et al, Virol.,141:221-234 (1985); Zimber et al, Virol., 196:900-904 (1993); andGregory et al, Nature, 349:612-614 (1991)). The cell lines includedAkata (type A, group I), Raji (type A, group III), P3HR1 (type B, groupI) (ATCC No. HTB-62), and Jijoye (type B, group III) (ATCC No. CCL-87).The molecules examined were a latent nuclear protein, EBNA2, a latentmembrane protein, LMP1, and an anti-apoptotic host component, bcl-2.Generally, group I cell lines exhibit negligible amounts of EBNA2, LMP1,and bcl-2, in contrast to group III lines that possess distinctivelylarge amounts of these molecules. These differences between the twophenotypes have been related to the sensitivity to induce apoptosis, andthe ability to induce the productive phase of EBV infection (Hendersonet al, Cell, 65:1107-1115 (1991); and Gregory et al, Nature, 349:612-614(1991)).

More specifically, the cells cultured in medium with or without 20 μg/mlvesnarinone for 7 days were harvested for Western blotting as describedabove. For detection of EBNA2, LMP1, and bcl-2, the mouse monoclonalanti-EBNA2 (PE2, Accurate, Westbury, N.Y.) (Young et al, N. Engl. J.Med., 321:1080-1085 (1989)), the mouse monoclonal anti-LMP1 (S12,provided by Dr. David A. Thorley-Lawson) (Mann et al, J. Virol.,55:710-720 (1985)), and the monoclonal anti-bcl-2 (bcl-2/124, Accurate)(Pezzella et al, Am. J. Pathol., 137:225-232 (1990)), respectively, wereused. The ECL chemiluminescence method was used for the detection ofthese proteins. Under these conditions, cell growth in the presence ofvesnarinone was reduced to 80-90% of the control cultures when cellnumbers were assessed. The results are shown in FIGS. 12A-12C.

As shown in FIGS. 12A-12C, vesnarinone did not induce these molecules inthe group I lines, nor did the vesnarinone alter levels of expression ofthese molecules in the group III lines. Thus, EBV-containing cellsexpressing different gene products, or susceptibility to apoptosis maybe differentially sensitive to vesnarinone.

G. Effects of Vesnarinone on Thymidine Kinase Activity in Anti-IgGTreated Akata Cells

It has been known that thymidine kinase plays an important role inmetabolic synthesis of dTTP (Sasayama et al, Heart vessels, 2:23-28(1986); and Feldman et al, Am. Heart J., 116:771-777 (1988)), and an EBVearly gene, BXLF1, encodes a thymidine kinase which is distinguishablefrom the cellular counterpart by a broad range of nucleoside ornucleobase analogues as substrates, and a relatively stronger resistanceto dTTP.

Thus, thymidine kinase activity was measured in Akata cells treated with50 μg/ml of anti-IgG which were cultured in the presence of vesnarinoneat a concentration of 100 μg/ml, and harvested 12 hrs later, accordingto the method of Turenne-Tessier et al, J. Virol., 57:1105-1112 (1986)with several modifications.

More specifically, 10⁷ cells were washed with PBS, and immediatelyfrozen with dry ice/ethanol. The pellet of frozen cells was suspended in100 μl of kinase extraction buffer comprising 50 mM Tris (pH 8.0), 50 mMKCl, 1.0 mM MgCl₂, 1.0 mM ATP, 1.0 mM DTT, 5.0% (v/v) glycerol, 80 μg/mlaprotinin, 40 μM leupeptin and 1.0 mM PMSF, and incubated on ice for 30min after a brief sonication. The nuclei were removed by high speedcentrifugation for 15 min and the supernatant (kinase extract) wasaliquoted and stored at -70° C. The protein concentration of the kinaseextract (about 4.0 mg/ml) was determined using standard Bradford Reagent(Bio-Rad Protein Assay Kit, Bio-Rad Laboratories, Hercules, Calif.),with BSA as a protein standard.

5.0 μl of the kinase extract was incubated at 37° C. with 35 μ1 ofkinase reaction buffer comprising 150 mM phosphate (pH 7.5), 20 mM ATP,20 mM MgCl₂, 40 mM KCl, 1.0 mM DTT, 10 mM NaF and 20 μM ³ H!thymidine(6.7 Ci/mmole, ICN) for 5 or 15 min. The reaction was terminated byplacing on ice, and an addition of 4.0 μl of 0.5M EDTA (pH 8.0). Formeasurement of dTTP-resistant thymidine kinase activity, a kinasereaction buffer additionally containing 100 μM dTTP (Pharmacia) wasused.

The amount of phosphorylated ³ H!thymidine derivatives formed during thereaction was determined by applying the reaction mixtures onto WhatmanDE-81 paper filters, which were then washed four times in 1.0 mMammoniumformate (pH 8.0), and once in ethanol before drying and liquidscintillation counting. An addition of vesnarinone into this in vitroassay system at concentrations up to 100 μg/ml, did not display anyinhibition of the thymidine kinase activity. The results are shown inFIG. 13A.

As shown in FIG. 13A, a high activity of whole thymidine kinase ingrowing cells was profoundly reduced in DMSO-arrested cells, andrestored to a small extent 13 hrs after release. A further additionalincrease in thymidine kinase activity was observed in cells treated withanti-IgG, and this increase was partially inhibited in the presence ofvesnarinone.

Next, thymidine kinase assays were performed in the same manner in thepresence of 100 μM dTTP, which preferentially suppresses the thymidinekinase of cellular origin. The results are shown in FIG. 13B.

As shown in FIG. 13B, there were high levels of dTTP-resistant thymidinekinase in anti-IgG treated cells. These observations indicate that theincrease in thymidine kinase activity observed in anti-IgG treated cellsis likely due to an expression of the EBV thymidine kinase gene (BXLF1).In addition, the increase in dTTP-resistant thymidine kinase was alsopartially suppressed by vesnarinone treatment.

As shown in this Example, vesnarinone inhibits the productive phase ofEBV infection in a cell system that offers a number of distinctadvantages. That is, following anti-IgG antibody treatment of Akatacells, there is a rapid induction of the productive viral cycle.Moreover, introduction of arrest of cell cycle progression followed byrelease, the treatment with anti-IgG antibody induces a more efficientand synchronous replication of EBV within 12 hrs, before the cells enterS-phase. This rapid and synchronous entry into the productive phaseenables one to elucidate events in a clearer fashion. Also, the eventscan be analyzed independently of those associated with host cellproliferation.

In this system, vesnarinone markedly inhibited the productivereplication of EBV genome in a dose-dependent manner. In contrast,little or no change was observed in the expression of the early genesassociated with exit from the latent stage, including ZEBRA, R and EA-R.Further, the broadened 2C peak of DNA detected by flow cytometry incells treated with anti-IgG antibody persisted in the presence ofvesnarinone. This configuration of DNA is evidence for entry into thelytic cycle. These data, which characterize stimulated entry into thelytic phase, appeared independently of the effect of vesnarinone onproductive replication of EBV genome detected by in situ hybridization.Only EA-D expression was markedly reduced by vesnarinone 20 hrs afteranti-IgG stimulation. EA-D is a co-factor of the EBV DNA polymerase, andis generally expressed at a later time point than the other three earlygenes. One possible explanation for this dissociation of effects onearly gene expression is that EA-D may be related to the EBV replicationunit (Kiehl et al, Virol., 184:330-340 (1991); and Daibata et al,Virol., 196:900-904 (1993)), and its expression may be regulated in partby the status of EBV DNA replication itself.

In the DMSO-released Akata cells, dTTP represented the smallestdeoxynucleobase pool, and was one of the major targets of vesnarinonetreatment. In addition, levels of dATP and dGTP were also influenced tothe same degree by vesnarinone treatment. There have been several linesof evidence linking depletion or alteration of deoxynucleobase poolswith reduction of DNA viral replication (Datta et al, Proc. Natl. Acad.Sci. USA, 77:5163-5166 (1980)), and an increase in the rate of mutationof reverse transcription of retroviruses (Vartanian et al, Proc. Natl.Acad. Sci. USA, 91:3092-3096 (1994)).

Anti-viral agents such as acyclovir, act as a competitor to dGTP in EBVDNA replication after modification to the active acyclovir triphosphate(Datta et al, Proc. Natl. Acad. Sci. USA, 77:5163-5166 (1980)). Thealterations in dTTP, dATP, and dGTP pool size by vesnarinone mayreasonably explain the suppression of EBV replication observed in thissystem. Among four dNTPs, dTTP was the only dNTP induced particularly bythe anti-IgG treatment, and this increase in dTTP level in the cellsseemed to be attributed to the expression of the EBV thymidine kinasewhich is recognized as an early gene product. The substantialsuppression of the increase in dTTP level in anti-IgG treated cells bytreatment with 100 μg/ml vesnarinone was comparable to the suppressionof induction of thymidine kinase activity observed under the sameculture conditions. The expression of this early gene (BXLF1) may beaffected by the treatment of this drug at this concentration similarlyto the case of ZEBRA.

These studies indicate that vesnarinone should prove to have therapeuticbenefit in disorders where reactivation of EBV is observed, and whereproductive replication of EBV plays an important role.

EXAMPLE 3 A. Effect of Vesnarinone on Inhibition of CMV Replication

Vesnarinone was evaluated for anti-CMV activity in vitro in a CMV plaquereduction assay.

More specifically, subconfluent monolayers of MRC₅ cells (Biowhittaker,Walkersville, Md.) in 12-well tissue culture plates were exposed to asuspension of a 1:500 dilution of stock virus (CMV strain AD169; ATCCNo. VR-538) in Eagles Minimal Essential Medium (MEM) supplemented with2.0% (v/v) fetal bovine serum, for 2 hr at 37° C. to allow for virusabsorption. The fluid containing unabsorbed virus was then removed, andthe cell layer rinsed with Eagles MEM without serum. Vesnarinone wasthen added to triplicate virus-infected cell cultures, in theconcentrations shown in Table 1 below, in 1.0 ml of overlay mediumcomprising Eagles MEM containing 2.0% (v/v) fetal bovine serum and 0.25%(w/v) agarose. The plates were then incubated at 37° C. in a humidifiedatmosphere containing 95% air/5% CO₂ until discrete foci of CPE(plaques) were formed at day 7. Plaque reduction resulting from theaddition of vesnarinone was evaluated by comparing the mean plaque countof vesnarinone-treated cultures with the mean plaque count in theuntreated virus-infected control cultures. The result are shown in Table1 below.

                  TABLE 1                                                         ______________________________________                                        Vesnarinone (μg/ml)                                                                      CMV Plaque Reduction (%)                                        ______________________________________                                        0             0                                                               3.88          10.69                                                           7.75          12.47                                                           15.5          19.27                                                           31            96.16                                                           ______________________________________                                    

As shown in Table 1 above, vesnarinone inhibits CMV plaque production ina dose-dependent manner. At the concentration of 30 μg/ml, which iswithin the therapeutic range, vesnarinone exhibited a very high level ofefficacy (91.5%) against CMV plaque formation.

B. Effect of Vesnarinone on Cytotoxicity of MRC₅ Cells

The cytotoxicity of vesnarinone on MRC₅ cells was determined bymicroscopic evaluation of the vesnarinone-treated cells described inSection A. above. A small amount of precipitate was observed at 31μg/ml. No cytotoxicity was observed.

The cytotoxicity of vesnarinone of MRC₅ cells was also determined byevaluating reduction of3-(4,5-dimethyl-thiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT) toMTT-formazan, by mitochondrial enzymes of viable cells solubilized bySDS, when measured spectrophotometrically as described by Moomann, J.Immunol. Methods, 65:55 (1983).

More specifically, 5.0 μg/ml of MTT in Eagles MEM supplemented with 2.0%(v/v) fetal bovine serum was added to each well, and incubated at 37° C.for 6 hr. The cells were then solubilized by adding 30% (w/v) SDS in0.02N HCl. The cultures were incubated overnight at 37° C. Then, theoptical density was measured at 570 nm on a spectrophotometer. Thecytotoxicity control was mock-infected cells exposed to medium only andoverlaid with vesnarinone in the concentrations shown in Table 2 below,which had been diluted in Eagles MEM containing 2.0% (v/v) fetal bovineserum. The absorbance (O.D. at 570 nm) of each vesnarinone-treated wellwas compared to the mean OD of the cell control wells. The cellviability, which is expressed as a percent of the control, is shown inTable 2 below.

                  TABLE 2                                                         ______________________________________                                        Vesnarinone  MTT Cell Viability                                               (μg/ml)   (% of control)                                                   ______________________________________                                        3.88         100                                                              7.75         98                                                               15.5         98                                                               31           90                                                               ______________________________________                                    

As shown in Table 2 above, little or no cytotoxicity was observed usingMTT to determine cell viability.

C. Effect of Vesnarinone and DHPG on Inhibition of CMV Replication

Vesnarinone and DHPG were evaluated for anti-CMV activity in vitro.

More specifically, vesnarinone, at the concentrations shown in Table 3below, was evaluated alone and in combination with DHPG, at theconcentrations shown in Table 3 below, in the CMV plaque reduction assaydescribed above. The percent inhibition of each compound andcombinations thereof are shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Vesnarinone                                                                             DHPG (μM)                                                        (μg/ml)                                                                              0        0.625  1.250   2.500 5.000                                 ______________________________________                                                CMV Plaque Reduction (%)                                              31.00     96.16    99.11  100     100   100                                   15.50     19.27    55.64  59.04   65.59 80.19                                 7.75      12.47    39.38  58.60   71.61 86.40                                 3.88      10.69    8.03   19.71   46.03 57.71                                 0         0        0      7.88    20.75 45.14                                 ______________________________________                                    

As shown in Table 3 above, vesnarinone reduced CMV plaque production by96.16% at the 31 μg/ml concentration. The other concentrations ofvesnarinone were only marginally active (19.27-10.69% reduction). DHPGat 5.0 μM, 2.5 μM and 1.25 μM showed 45.14, 20.75 and 7.88% reduction ofCMV plaques. The compound combinations showed various degrees of % CMVplaque reduction.

The minimum compound concentration that reduces plaque production by 50%(MIC₅₀) was calculated from single compound assays by using a regressionanalysis program for semilog curve fitting. The MIC₅₀ for vesnarinonewas 24 μg/ml, and for DHPG it was >5.0 μM.

The above data was then analyzed using the technique described byPrichard et al, Antiviral Res., 14:181-206 (1990), which involves adetermination of the difference between the observed combined effectsand those expected if the interactions of the two compounds were simplyadditive. The individual effects of the compounds were used to calculatethe additive effects, and these were subtracted from the observedcombined effects. The following formula of additivity, which isappropriate for inhibitors affecting different sites, was used.

    Z=X+Y (1-X)

where Z is total inhibition (activity); X is the activity observed witha specified concentration of compound X; and Y (1-X) is the activity ofcompound Y when only part of the activity remains to be inhibited(Prichard et al, supra).

After additive effects were subtracted from observed combined effects,positive values or activity beyond the additive effects were suggestiveof synergy. Negative values or activity below the additive effects weresuggestive of antagonism. The results are shown in Table 4 below.

                  TABLE 4                                                         ______________________________________                                        Vesnarinone                                                                             DHPG (μM)                                                        (μg/ml)                                                                              0        0.625  1.250    2.500                                                                              5.000                                 ______________________________________                                                CMV Plaque Reduction (%)                                              31.00     0        0      3.54     3.04 2.11                                  15.50     0        13.83  15.27    21.65                                                                              8.20                                  7.75      0        13.89  22.23    35.68                                                                              22.70                                 3.88      0        0      0        0    0.59                                  0         0        0      0        0    0                                     ______________________________________                                    

As shown in Table 4 above, at the 95% confidence level, there wassignificant synergy. The synergy peak was seen at a combination of 2.5μM of DHPG and 7.75 μg/ml of vesnarinone. In general, there was a bandof synergy involving the 15.5 μg/ml and 7.75 μg/ml of vesnarinone, whencombined with all doses of DHPG.

Thus, vesnarinone had anti-CMV activity, and such enhanced theanti-viral activity of DHPG. Based on the Prichard et al, supra,technique for evaluating compound combinations, it is believed that themajor interaction of vesnarinone and DHPG is synergistic.

D. Effect of Vesnarinone and DHPG on Cytotoxicity of MRC₅ Cells

Cytotoxicity was determined by microscopic examination of thecompound-treated mock-infected cell controls, and also by evaluating thereduction of MTT, by mitochondrial enzymes of viable cells, toMTT-formazan, as described above. A small amount of precipitate wasobserved at 31 μg/ml of vesnarinone, and all drug combination (DHPG)doses using 31 μg/ml of vesnarinone. The results are shown in Table 5below.

                  TABLE 5                                                         ______________________________________                                        Vesnarinone                                                                             DHPG (μM)                                                        (μg/ml)                                                                            0          0.625  1.250    2.500                                                                              5.000                                 ______________________________________                                                MTT Cell Viability (% of control)                                     31.00   90          87     84       85   90                                   15.50   98          99     95       96   98                                   7.75    98         100    100      100  100                                   3.88    100        100    100      100  100                                   0        0         100    100      100  100                                   ______________________________________                                    

As shown in Table 5 above, little or no cytotoxicity was observed withMTT to determine cell viability, nor was such observed microscopically.

EXAMPLE 4 A. Effect of Vesnarinone on Thymidine and AZT Uptake

AZT is more lipophilic than the physiological nucleosides. Thus, AZT isexpected to bypass the nucleoside transporter, and enter cells bypassive diffusion. Dipyridamole, a specific inhibitor of nucleosidetransport, is known to inhibit transport of physiological nucleosides,but not AZT. Thus, the effect of the nucleoside transport inhibitor,vesnarinone on the uptake of ³ H!AZT was examined using the rapid uptakemethod described by Aronow et al, J. Biol. Chem., 260:16274-16278(1985).

More specifically, 2.0×10⁶ CHO cells or PHA-activated human T cells wereresuspended in 100 μl of RPMI 1640 medium, supplemented with 10.0% (v/v)heat-inactivated fetal bovine serum and 2.0 mM L-glutamine, 100 U/ml ofpenicillin and 100 μg/ml of streptomycin, and pre-incubated for 20 minat 37° C. in the presence of 0-250 μM vesnarinone.

Human PBMC were obtained by leukopheresis of HIV-1 and HBV Zeronegativehealthy donors. Mononuclear cell suspensions were prepared therefrom byFicoll-Hypaque gradient centrifugation, and the T cells were obtainedtherefrom by E-rosette enrichment as described by Terada et al, J.Immunol., 147:698-704 (1991).

Vesnarinone was prepared as a stock solution containing 10 mg ofvesnarinone dissolved in 0.5 ml of 2.0N HCl, which was then added to 9.0ml of RPMI 1640 medium supplemented with 20 mM HEPES buffer (pH 7.55),and neutralized to pH 7.0 by addition of 2.0N NaOH. The resultingneutralized vesnarinone solution (1.0 mg/ml) was immediately filteredusing a 0.45 μm Millipore filter, and added to the cell cultures in theamounts shown in FIGS. 14A and 14B.

Then, 1.0 μCi/ml of ³ H!thymidine (6.7 Ci/mmol) or 1.0 μCi/ml of ³ H!AZT(14 Ci/mmol) was added, and the mixture was immediately layered onsilicone oil/paraffin oil (96:6) in microfuge tubes. After 1 min, thecells were separated from free nucleoside or nucleobase in the waterphase, by centrifugation at 14,000 rpm (10,000×g) for 2 min. The waterand oil phases were discarded, and radioactivity of the cell pellets wasmeasured using liquid scintillation counting. ³ H!substrate uptake wasevaluated as a percentage of the control, i.e., cells treated with thesolution used in preparing the stock vesnarinone alone. The results areshown in FIGS. 14A and 14B. As shown in FIGS. 14A and 14B, vesnarinoneinhibits the uptake of ³ H!thymidine in a dose-dependent manner in CHOcells (FIG. 14A), and in primary human T lymphocytes (FIG. 14B), aspreviously reported (Kumakura et al, Life Sciences, 57:PL57-82 (1995)).In contrast, the uptake of ³ H!AZT was not affected by vesnarinone.Similar results were obtained using the human monoblastic cell line,U937 (ATCC No. CRL-1593), and Raji cells. Thus, like dipyridamole,vesnarinone is a specific inhibitor of thymidine uptake, and not of AZTuptake.

B. Phosphorylation of AZT in the Presence of Vesnarinone

Differential inhibition by vesnarinone of thymidine and AZT entry in thecell would be expected to suppress any antagonistic influence ofthymidine on the intracellular phosphorylation of AZT. This wouldprovide a therapeutic potential for the combination therapy ofvesnarinone with AZT to potentiate the activity of the latter in cells.The activity of AZT in inhibiting viral replication of retroviruses suchas HIV-1, is based on achieving increased concentrations ofphosphorylated AZT in the cells (Weinstein et al, Ann. NY Acad. Sci.,616:367-384 (1991)). Thus, to determine if inhibition of thymidineuptake by vesnarinone has any influence on intracellular phosphorylationof AZT, the effect of vesnarinone on intracellular accumulation ofphosphorylated forms of ³ H!AZT was evaluated.

More specifically, 1.0×10⁷ PHA-activated human T lymphocytes weresuspended in 1.0 ml of RPMI 1640 medium supplemented with 10.0% (v/v)heat-inactivated fetal bovine serum and 2.0 mM L-glutamine, 100 U/ml ofpenicillin and 100 μg/ml of streptomycin, in the presence of 0, 75 or250 μM vesnarinone. 10 μCi/ml of ³ H!thymidine (6.7 Ci/mmole) or 10μCi/ml of ³ H!AZT (14 Ci/mmole) were added, and the mixture wasincubated at 37° for 0, 30, 60 and 120 min. The cells were thenharvested, washed with PBS, and lysed in 400 l of 65% (v/v) ethanol for20 min at 0° C. The supernatant was separated by centrifugation at14,000 rpm (10,000×g) for 5 min, and applied to DEAE filter paper(Whatman DE-81). The filter paper was washed four times with 1.0 mMammonium formate for 10 min each, and the radioactivity was measuredusing liquid scintillation. The results are shown in FIGS. 15A and 15B.As shown in FIG. 15B, vesnarinone augmented the accumulation ofphosphorylated AZT in PHA-activated human T lymphocytes. In contrast, asshown in FIG. 15A, vesnarinone delayed the accumulation ofphosphorylated ³ H!thymidine in cellular extracts after removal ofchromosomal debris. Similar results were obtained when using U937 cells,in place of the PHA-activated human T lymphocytes.

C. Phosphorylated Forms of AZT

AZT-TP (AZT-triphosphate) is an effective form mis-utilized by viralreverse transcriptase (Weinstein et al, supra). Thus, the effect ofvesnarinone on intracellular concentrations of AZT-MP, AZT-DP, andAZT-TP in U937 cells was next examined.

More specifically, 1.0×10⁷ /ml U937 cells in 1.0 ml of RPMI 1640 medium,supplemented with 10.0% (v/v) heat-inactivated fetal bovine serum and2.0 mM L-glutamine, 100 U/ml of penicillin and 100 μg/ml ofstreptomycin, were incubated at 37° C. with 5.0 μCi/ml of ³ H!thymidine(6.7 Ci/mmol) or 5.0 μCi/ml of ³ H!AZT (14 Ci/mmol) for 2 hr. The cellswere then harvested, washed with PBS, and lysed in 400 μl of 65% (v/v)ethanol for 20 min at 0° C. The supernatant was separated bycentrifugation at 14,000 rpm (10,000×g) for 5 min, and subjected to HPLCusing an anion exchange column (SAX, Phenomenex). The concentration ofKH₂ PO₄ (pH 4.1) in the column was linearly increased from 15 mM to 1.0Min 55 min at a flow rate of 1.0 ml/min. 1.0 ml fractions were collected,and the radioactivity was measured by liquid scintillation. Theretention time for AZT and AZT-MP was confirmed using cold AZT andAZT-MP (Sigma) under the same conditions. The results are shown in FIGS.16A and 16B. As shown in FIG. 16A, thymidine was effectivelyphosphorylated up to the triphosphate level in cells. In contrast, asshown in FIG. 16B, AZT was phosphorylated to AZT-MP, but poorlyphosphorylated beyond this stage to the diphosphate form. This indicatedthat AZT is a good substrate for thymidine kinase, but not forthymidylate kinase in cells.

D. Concentration of Phosphorylated Forms of AZT

Next, the effect of vesnarinone on intracellular concentrations ofAZT-MP, AZT-DP and AZT-TP was examined.

More specifically, 1.0×10⁷ /ml U937 cells in 1.0 ml of RPMI 1640 medium,supplemented with 10.0% (v/v) heat-inactivated fetal bovine serum and2.0 mM L-glutamine, 100 U/ml of penicillin and 100 μg/ml ofstreptomycin, were incubated with 5.0 μCi/ml of ³ H!AZT (14 Ci/mmol) for2 hr at 37° C. in the presence of 0, 12.5, 25 or 75 μM vesnarinone.Intracellular concentrations of ³ H!AZT-MP, ³ H!AZT-DP, and ³ H!AZT-TPwere measured using HPLC as described above. The results, which arerepresented by a ratio of sample ³ H!AZT-NP (cpm)/control ³ H!AZT-NP(cpm), are shown in FIG. 17. As shown in FIG. 17, vesnarinone, at aconcentration of 75 μM (30 μg/ml), increased intracellular levels ofAZT-MP, AZT-DP, and AZT-MP by 100%, 45% and 25%, respectively. Theseresults indicate that vesnarinone increases the intracellular effectiveform of AZT when the two compounds are added together.

E. Phosphorylation of Thymidine Vesnarinone vs. Dipyridamole

Dipyridamole is a powerful inhibitor of nucleoside transport, and isalso expected to enhance the biological activities of AZT in retroviralinfection (Weinstein et al, supra). Thus, the comparative abilities ofvesnarinone and dipyridamole to potentiate phosphorylation of thymidinewere examined.

More specifically, the uptake of ³ H!thymidine in U937 cells, within 1min in the presence of 0-250 μM vesnarinone or 0-1000 nM dipyridamolewas measured as described above. Dipyridamole was prepared as a 50 mMstock solution dissolved in ethanol. The results are shown in FIGS. 18Aand 18B. As shown in FIGS. 18A and 18B, both vesnarinone (FIG. 18A) anddypridamole (FIG. 18B) inhibited the uptake of thymidine in adose-dependent manner. FIGS. 18A and 18B also demonstrate thatdipyridamole is a more potent inhibitor of nucleoside transport thanvesnarinone; the IC₅₀ for thymidine uptake was <10 nM in dipyridamole,and -25 μM in vesnarinone.

F. Phosphorylation of AZT Vesnarinone vs. Dipyridamole

As discussed above, neither 0-250 μM vesnarinone nor 0-1000 nMdipyridamole have an effect on thymidine uptake, indicating that both ofthem are selective inhibitors of physiological thymidine uptake. Thus,the effects of these compounds on intracellular phosphorylated AZT wasthen examined.

More specifically, 1.0×10⁷ /ml U937 cells in 1.0 ml RPMI 1640 medium,supplemented with 10.0% (v/v) heat-inactivated fetal bovine serum and2.0 mM L-glutamine, 100 U/ml of penicillin and 100 μg/ml ofstreptomycin, were incubated with 5.0 μCi/ml of ³ H!AZT (14 Ci/mmol) at37° C. for 2 hr in the presence of 0-250-μM vesnarinone or 0-100 nMdipyridamole. The cells were then harvested, washed with PBS, and lysedin 400 μl of 65% (v/v) ethanol for 20 min at 0° C. The supernatant wasseparated by centrifugation at 14,000 rpm (10,000×g) for 5 min, andapplied to DEAE filter paper (Whatman DE-81). The filter paper waswashed four times with 1.0 mM ammonium formate for 10 min each, and theradioactivity was measured using liquid scintillation. The results areshown in FIGS. 19A and 19B. As shown in FIG. 19A, vesnarinone increasedthe concentration of phosphorylated AZT in U937 cells in adose-dependent manner. However, as shown in FIG. 19B, surprisingly 0-100nM dipyridamole, which inhibits thymidine transport to a similar extentas 0-250 μM vesnarinone, did not affect intracellular concentrations ofphosphorylated AZT. This is consistent with the findings previouslyreported for AZT (Weinstein et al, supra). Although the mechanism(s)underlying this differential effect remains unclear at present, it isimportant to note the differences between these two compounds asnucleoside/nucleobase transport inhibitors (Kumakura et al, supra).Vesnarinone inhibits both nucleoside and nucleobase transport in asimilar manner, whereas dipyridamole selectively inhibits nucleosidetransport without affecting nucleobase transport at clinicallyachievable doses (<10 μM) (Kumakura et al, supra). In fact, highconcentrations of dipyridamole increase phosphorylation of AZT as wellas vesnarinone. These results suggest that the mechanism resulting indifferential inhibition of nucleoside/nucleobase transport might becorrelated with the differential phosphorylation of AZT. Alternatively,dipyridamole or vesnarinone may have another activity, decreasing orincreasing phosphorylation of AZT in cells, independently of nucleosidetransport inhibition.

G. Enhanced Anti-HIV-1 Effect

Although the mechanism to render the above-discussed differential effectof vesnarinone remains undetermined to date, the data suggest thatvesnarinone may be more effective in enhancing the anti-viral activityof AZT than dipyridamole by increasing intracellular concentrations ofAZT-TP. Thus, potentiation of the anti-HIV-1 effect of AZT byvesnarinone was investigated by evaluating responses of two independentstrains of HIV-1 isolated from clinical samples, one of themAZT-sensitive (18a), the other AZT-resistant (18c).

More specifically, PBMC were incubated for 48 hr with 3.0 μg/ml of PHAand 10 U/ml of IL-2. Then, 10-100 TCID₅₀ virus/ml of clinical isolatestrains of HIV-1, 18a (AZT-sensitive) or 18c (AZT-resistant), wereadded. 2 hr after infection, the cells were separated from the virus bycentrifugation, washed twice with culture medium, and incubated in96-well culture plates in the presence or absence of vesnarinone and/orAZT in the concentrations shown in FIGS. 20A and 20B. After 5 days ofinfection, the supernatant was collected and HIV-1 p24 antigen wasmeasured using the p24 ELISA kit (NEN Dupont).

The combination index (CI) was calculated using multiple drug effectanalysis of Chou and Tala, as described by Johnson et al, J. Infect.Dis., 159:837-844 (1989). CI values of <1, =1, and >1, indicatesynergism, additive effects, and antagonism, respectively. The resultsare shown in FIG. 20A (strain 18a:AZT-sensitive) and FIG. 20B (strain18c:AZT-resistant). As shown in FIG. 20A, AZT alone inhibited p24production in 18a infected cells (IC₅₀ =0.67 nM). In contrast, as shownin FIG. 20B, AZT alone did not inhibit p24 production in 18c infectedcells (IC₅₀ >640 nM). Vesnarinone by itself also had an inhibitoryeffect on p24 production, but only at the high concentrations (>50˜100μg/ml). As demonstrated in FIG. 20A and 20B, vesnarinone, atconcentrations achievable in the serum, potentiated the reduction of p24by AZT on both 18a and 18c infected cells. The combination indices inthis and other experiments were between 0.005 and 0.13 for 18a and 0.06and 0.59 for 18c. These results indicate that vesnarinone potentiatesthe anti-HIV-1 activity of AZT in these cells.

In summary, Example 4 demonstrates that (1) vesnarinone inhibitstransport of thymidine but not of AZT; (2) vesnarinone enhancesphosphorylation of AZT in cells; and that (3) vesnarinone potentiatesanti-HIV-1 activity of AZT in vitro. These results indicate that thecombination of vesnarinone and AZT should be useful for patientsinfected with HIV.

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

What is claimed:
 1. A method for inhibiting DNA virus replication,comprising exposing cells which have been infected with said DNA virusto a pharmaceutically effective amount of a carbostyril derivativerepresented by the following general formula (1), or a pharmaceuticallyacceptable salt thereof: ##STR3## wherein R is a benzoyl group which mayoptionally have lower alkoxy groups on the phenyl ring as substituentsand the carbon-carbon bond in the 3 and 4 positions of the carbostyrilskeleton is a single bond or double bond.
 2. The method of claim 1,wherein said carbostyril is 3,4-dihydro-6-4-(3,4-dimethoxybenzoyl)-1-piperazinyl!-2(1H)-quinoline or apharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein said DNA virus is selected from the group consisting of herpessimplex virus type 1, herpes simplex virus type 2, human herpes virustype 6, herpes zoster virus, human cytomegalovirus and Epstein-Barrvirus.
 4. The method of claim 3, wherein said DNA virus is Epstein-Barrvirus.
 5. The method of claim 3, wherein said DNA virus iscytomegalovirus.
 6. A method for treating Epstein-Barr virus infection,comprising administering to a subject in need of such treatment, apharmaceutically effective amount of a carbostyril derivativerepresented by the following general formula (1), or a pharmaceuticallyacceptable salt thereof: ##STR4## wherein R is a benzoyl group which mayoptionally have lower alkoxy groups on the phenyl ring as substituentsand the carbon--carbon bond in the 3 and 4 positions of the carbostyrilskeleton is a single bond or double bond.
 7. The method of claim 6,wherein said carbostyril is 3,4-dihydro-6-4-(3,4-dimethoxybenzoyl)-1-piperazinyl!-2(1H)-quinoline or apharmaceutically acceptable salt thereof.
 8. The method of claim 6,wherein said carbostryril is co-administered with an anti-DNA viruscompound selected from the group consisting of 9-(2-hydroxyethoxy)methyl!guanine and 9-(1,3-hydroxy-2-propoxymethyl)guanine.
 9. A method for treating cytomegalovirus infection, comprisingadministering to a subject in need of such treatment, a pharmaceuticallyeffective amount of a carbostyril derivative represented by thefollowing general formula (1), or a pharmaceutically acceptable saltthereof: ##STR5## wherein R is a benzoyl group which may optionally havelower alkoxy groups on the phenyl ring as substituents and thecarbon-carbon bond in the 3 and 4 positions of the carbostyril skeletonis a single bond or double bond.
 10. The method of claim 9, wherein saidcarbostyril is 3,4-dihydro-6-4-(3,4-dimethoxybenzoyl)-1-piperazinyl!-2(1H)-quinoline or apharmaceutically acceptable salt thereof.
 11. The method of claim 9,wherein said carbostryril is co-administered with9-(1,3-dihydroxy-2-propoxymethyl) guanine.
 12. A method for inhibitingRNA virus replication, comprising exposing cells which have beeninfected with said RNA virus to a pharmaceutically effective amount ofan anti-RNA virus compound and a pharmaceutically effective amount of acarbostyril derivative represented by the following general formula (1),or a pharmaceutically acceptable salt thereof: ##STR6## wherein R is abenzoyl group which may optionally have lower alkoxy groups on thephenyl ring as substituents and the carbon-carbon bond in the 3 and 4positions of the carbostyril skeleton is a single bond or double bond.13. The method of claim 12, wherein said carbostyril is 3,4-dihydro-6-4-(3,4-dimethoxybenzoyl)-1-piperazinyl!-2(1H)-quinoline or apharmaceutically acceptable salt thereof.
 14. The method of claim 12,wherein said anti-RNA virus compound is selected from the groupconsisting of 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine,2'3'-dideoxyinosine, 2'3'-didehydro-2'3'-dideoxythymidine and5'3'-dideoxythiacytidine.
 15. The method of claim 12, wherein said RNAvirus is selected from the group consisting of HIV, adult T-cellleukemia virus, and human immune deficiency virus type II.
 16. Themethod of claim 15, wherein said RNA virus is HIV.
 17. A method fortreating HIV virus infection, comprising administering to a subject inneed of such treatment, a pharmaceutically effective amount of ananti-HIV virus compound and a pharmaceutically effective amount of acarbostyril derivative represented by the following general formula (1),or a pharmaceutically acceptable salt thereof: ##STR7## wherein R is abenzoyl group which may optionally have lower alkoxy groups on thephenyl ring as substituents and the carbon-carbon bond in the 3 and 4positions of the carbostyril skeleton is a single bond or double bond.18. The method of claim 17, wherein said carbostyril is 3,4-dihydro-6-4-(3,4-dimethoxybenzoyl)-1-piperazinyl!-2(1H)-quinoline or apharmaceutically acceptable salt thereof.
 19. The method of claim 17,wherein said anti-HIV virus compound is selected from the groupconsisting of 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine,2'3'-dideoxyinosine, 2'3'-didehydro-2'3'-dideoxythymidine and5'3'-dideoxythiacytidine.
 20. A method for augmenting phosphorylation ona nucleoside analogue comprising co-administering to cells, saidnucleoside analogue and a pharmaceutically effective amount of acarbostyril derivative represented by the following general formula (1),or a pharmaceutically acceptable salt thereof: ##STR8## wherein R is abenzoyl group which may optionally have lower alkoxy groups on thephenyl ring as substituents and the carbon-carbon bond in the 3 and 4positions of the carbostyril skeleton is a single bond or double bond.21. The method of claim 20, wherein said carbostyril is 3,4-dihydro-6-4-(3,4-dimethoxybenzoyl)-1-piperazinyl!-2(1H)-quinoline or apharmaceutically acceptable salt thereof.
 22. The method of claim 20,wherein said nucleoside analogue is selected from the group consistingof 9- (2-hydroxyethoxy)methyl! guanine, 9-(1,3-hydroxy-2-propoxymethyl)guanine, 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine,2'3'-dideoxyinosine, 2'3'-didehydro-2'3'-dideoxythymidine and5'3'-dideoxythiacytidine.